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MITF activity is regulated by a direct interaction with RAF proteins in melanoma cells.

ABSTRACT: The MITF transcription factor and the RAS/RAF/MEK/ERK pathway are two interconnected main players in melanoma. Understanding how MITF activity is regulated represents a key question since its dynamic modulation is involved in the phenotypic plasticity of melanoma cells and their resistance to therapy. By investigating the role of ARAF in NRAS-driven mouse melanoma through mass spectrometry experiments followed by a functional siRNA-based screen, we unexpectedly identified MITF as a direct ARAF partner. Interestingly, this interaction is conserved among the RAF protein kinase family since BRAF/MITF and CRAF/MITF complexes were also observed in the cytosol of NRAS-mutated mouse melanoma cells. The interaction occurs through the kinase domain of RAF proteins. Importantly, endogenous BRAF/MITF complexes were also detected in BRAF-mutated human melanoma cells. RAF/MITF complexes modulate MITF nuclear localization by inducing an accumulation of MITF in the cytoplasm, thus negatively controlling its transcriptional activity. Taken together, our study highlights a new level of regulation between two major mediators of melanoma progression, MITF and the MAPK/ERK pathway, which appears more complex than previously anticipated.

SUBMITTER: Estrada C

PROVIDER: S-EPMC8799692 | biostudies-literature | 2022 Jan

REPOSITORIES: biostudies-literature

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MITF activity is regulated by a direct interaction with RAF proteins in melanoma cells.

Estrada Charlène C Mirabal-Ortega Liliana L Méry Laurence L Dingli Florent F Besse Laetitia L Messaoudi Cedric C Loew Damarys D Pouponnot Celio C Bertolotto Corine C Eychène Alain A Druillennec Sabine S

Communications biology 20220128 1

The MITF transcription factor and the RAS/RAF/MEK/ERK pathway are two interconnected main players in melanoma. Understanding how MITF activity is regulated represents a key question since its dynamic modulation is involved in the phenotypic plasticity of melanoma cells and their resistance to therapy. By investigating the role of ARAF in NRAS-driven mouse melanoma through mass spectrometry experiments followed by a functional siRNA-based screen, we unexpectedly identified MITF as a direct ARAF p ...[more]

PMID: 35091687

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Project description:BackgroundMelanoma is a cancer with rising incidence and new therapeutics are needed. For this, it is necessary to understand the molecular mechanisms of melanoma development and progression. Melanoma differs from other cancers by its ability to produce the pigment melanin via melanogenesis; this biosynthesis is essentially regulated by microphthalmia-associated transcription factor (MITF). MITF regulates various processes such as cell cycling and differentiation. MITF shows an ambivalent role, since high levels inhibit cell proliferation and low levels promote invasion. Hence, well-balanced MITF homeostasis is important for the progression and spread of melanoma. Therefore, it is difficult to use MITF itself for targeted therapy, but elucidating its complex regulation may lead to a promising melanoma-cell specific therapy.MethodWe systematically analyzed the regulation of MITF with a novel established transcription factor based gene regulatory network model. Starting from comparative transcriptomics analysis using data from cells originating from nine different tumors and a melanoma cell dataset, we predicted the transcriptional regulators of MITF employing ChIP binding information from a comprehensive set of databases. The most striking regulators were experimentally validated by functional assays and an MITF-promoter reporter assay. Finally, we analyzed the impact of the expression of the identified regulators on clinically relevant parameters of melanoma, i.e. the thickness of primary tumors and patient overall survival.ResultsOur model predictions identified SOX10 and SOX5 as regulators of MITF. We experimentally confirmed the role of the already well-known regulator SOX10. Additionally, we found that SOX5 knockdown led to MITF up-regulation in melanoma cells, while double knockdown with SOX10 showed a rescue effect; both effects were validated by reporter assays. Regarding clinical samples, SOX5 expression was distinctively up-regulated in metastatic compared to primary melanoma. In contrast, survival analysis of melanoma patients with predominantly metastatic disease revealed that low SOX5 levels were associated with a poor prognosis.ConclusionMITF regulation by SOX5 has been shown only in murine cells, but not yet in human melanoma cells. SOX5 has a strong inhibitory effect on MITF expression and seems to have a decisive clinical impact on melanoma during tumor progression.

Dataset Information

MITF activity is regulated by a direct interaction with RAF proteins in melanoma cells.

Publications

MITF activity is regulated by a direct interaction with RAF proteins in melanoma cells.

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